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WO1984001150A1 - Hormone de croissance d'aviens - Google Patents

Hormone de croissance d'aviens Download PDF

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Publication number
WO1984001150A1
WO1984001150A1 PCT/US1983/001388 US8301388W WO8401150A1 WO 1984001150 A1 WO1984001150 A1 WO 1984001150A1 US 8301388 W US8301388 W US 8301388W WO 8401150 A1 WO8401150 A1 WO 8401150A1
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WIPO (PCT)
Prior art keywords
leu
ctg
glu
thr
lys
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PCT/US1983/001388
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English (en)
Inventor
Lawrence M Souza
Thomas C Boone
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Amgen Inc
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Amgen Inc
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Priority to JP83503097A priority Critical patent/JPS59501852A/ja
Publication of WO1984001150A1 publication Critical patent/WO1984001150A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C12N15/71—Expression systems using regulatory sequences derived from the trp-operon
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/184—Hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C12N15/69—Increasing the copy number of the vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the present invention relates generally to the manipulation of genetic materials and, more particu ⁇ larly to the use of recombinant procedures to secure the production of polypeptides possessing one or more bio ⁇ chemical and immunological properties of growth hor ⁇ mones of avian species.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acids
  • the repeating units in DNA polymers are four different nucleotides, each of which consists of either a purine (adenine or guanine) or a pyrimidine (thymine or cytosine) bound to a deoxy- ribose sugar to which a phosphate group is attached.
  • nucleotides in linear polymeric form is by means of fusion of the 5' phosphate of one nucleotide to the 3* hydroxyl group of another.
  • Functional DNA occurs in the form of stable double stranded associations of single strands of nucleotides (known as deoxyoligo- nucleotides) , which associations occur by means of hydrogen bonding between purine and pyrimidine bases [i.e., ⁇ complementary*' associations existing either between adenine (A) and thymine (T) or guanine (G) and cytosine . (C) 1.
  • nucleotides are referred to by the names of their constituent purine or pyrimidine bases, and the complementary associations of nucleotides in double stranded DNA (i.e., A-T and G-C) are referred to as "base pairs".
  • Ribonucleic acid is a polynucleotide comprising adenine, guanine, cytosine and uracil (U) , rather than thymine, bound to ribose and a phosphate group.
  • DNA is generally effected through a process wherein specific DNA nucleotide sequences (genes) are "tran ⁇ scribed” into relatively unstable messenger RNA (RNA) polymers.
  • RNA messenger RNA
  • the mRNA serves as a template for the formation of structural, regulatory and catalytic proteins from amino acids. This mRNA ⁇ translation" process involves the operations of small RNA strands
  • tRNA transport and align individual amino acids along the mRNA strand to allow for formation of poly- peptides in proper amino acid sequences.
  • the mRNA "message”, derived from DNA and providing the basis for the tRNA supply and orientation of any given one of the twenty amino acids for polypeptide "expression”, is in the form of triplet "codons” — sequential groupings of three nucleotide bases. In one sense, the formation of a protein is the ultimate form of "expression" of the ⁇ r.
  • Promoter DNA sequences usually "precede” a gene in a DNA polymer and provide a site for initiation of the transcription into mRNA.
  • "Regulator” DNA se ⁇ quences also usually “upstream” of (i.e., preceding) a gene in a given DNA polymer, bind proteins that determine the frequency (or rate) of transcription initiation.
  • promoter/regulator or "control” DNA sequence, these sequences which precede a selected gene (or series of genes) in a functional DNA polymer cooperate to determine whether the transcription (and eventual expression) of a gene will occur.
  • DNA sequences which "follow" a gene in a DNA polymer and provide a signal for termination of the transcription into mRNA are referred to as transcription "terminator" sequences.
  • a focus of microbiological processing for nearly the last decade has been the attempt to manufac- ture industrially and pharmaceutically significant sub ⁇ stances using organisms which do not initially have genetically coded information concerning the desired product included in their DNA.
  • a gene that specifies the structure of a product is either isolated from a "donor” organism or chemically synthesized and then stably introduced into another organism which is preferably a self-replicating unicellular microorganism. Once this is done, the existing machinery for gene expres ⁇ sion in the "transformed" host cells operates to con- struct the desired product.
  • Selected foreign ("exogenous” or heterologous”) DNA strands are also prepared in linear form through use of similar enzymes.
  • the linear viral or plasmid DNA is incubated with the foreign DNA in the presence of li- gating enzymes capable of effecting a restoration process and "hybrid" vectors are formed which include the se ⁇ lected foreign DNA segment "spliced" into the viral or circular DNA plasmid. Transformation of compatible unicellular host organisms with the hybrid vector results in the formation of multiple copies of the foreign DNA in the host cell population. In some instances, the desired result is simply the amplification of the foreign DNA and the "product" harvested is DNA.
  • the goal of transformation is the expression by the host cells of the foreign DNA in the form of large scale synthesis of isolatable quantities of commercially significant pro ⁇ tein or polypeptide fragments coded for by the foreign DNA. See also, e.g., U.S. Letters Patent Nos. 4,269,731
  • restriction endonuclease enzymes are currently commercially available [See, e.g., "BRL Restriction Endonuclease Reference Chart" appearing in the " * 81/'82 Catalog” of Bethesda Research Laboratories, Inc., Gaithersburg, Maryland] . Verification of hybrid forma ⁇ tion is facilitated by chromatographic techniques which can, for example, distinguish the hybrid plasmids from non-hybrids on the basis of molecular weight. Other useful verification techniques involve radioactive DNA hybridization.
  • the gene to be inserted in a DNA vector must either be fitted with a new, host-accommodated tran- scription and translation regulating DNA sequence prior to insertion or it must be inserted at a site where it will come under the control of existing transcription and translation signals in the vector DNA.
  • growth hormone and "somatotropin” are generically employed to designate hormonally active polypeptides secreted by the anterior lobe of pituitary glands of a variety of vertebrate species. In all spe ⁇ cies, the growth hormones function generally to regulate the rate of skeletal growth and gain in body weight. Hypophyseal dysfunction and consequent variations in growth hormone production have been associated with growth abnormalities such as giantism and dwarfism.
  • bovine growth hormone bGH
  • the complete, 191 amino acid sequence of bGH has been determined and recombinant methods have been em ⁇ ployed to clone and sequence cDNA sequences coding for bGH and its precursor polypeptide form which includes a 26 amino acid "signal" or "leader” sequence which is cellularly deleted prior to entry of the hormone into circulation. See, e.g. , Miller, et al. , J.Biol.Chem. , 255, pp. 7521-7523 (1980).
  • rat growth hormone rat growth hormone
  • the prediction takes on aspects of scientifically-determined fact.
  • complete "verification" of the predicted polypeptide sequence as identical to that of the previously incompletely sequenced native sub ⁇ stance will ordinarily be had upon finding that the poly ⁇ peptide product of microbial expression of the cDNA possesses the biochemical and immunological properties of the native substance.
  • avian growth hormone indi ⁇ cate that a high plasma level of growth hormone in young birds is at least partially responsible for their high growth rate.
  • the secretion of growth hormone in young chickens is found to be very unstable and affected by stressful stimuli. Normally high circulating concentra ⁇ tions of growth hormone in young chickens are depressed by anesthesia, cold stress, infection, and certain other hormones, the release of which would be expected to be increased during stress.
  • the plasma concentration of growth hormone in young birds is also affected by meta ⁇ bolic and nutritional factors. Under conditions of nutritional deprivation, growth hormone exerts a meta ⁇ bolic role, decreasing energy flow into lipids.
  • avian growth hormones are pres ⁇ ently conducted by fractional purification of extracts from either pituitaries of the avian species, or from avian plasma containing circulating growth hormone. Both procedures provide very small amounts of impure polypep ⁇ tide substances.
  • Avian growth hormones, including growth hormone specifically isolatable from domesticated chickens, have not heretofore been completely sequenced and, even if the sequence were completely determined, the application of the presently available methods for the chemical synthesis of entire polypeptides of nearly two hundred amino acids would likely provide exceedingly low yields at very high cost.
  • recombi- nant DNA techniques have for the first time been brought to bear to effect the quantitative microbial synthesis of polypeptide substances having the biochemical and immunological properties of avian growth hormones.
  • the present invention provides polypeptides characterized by in ⁇ cluding part or all of the following amino acid sequence (commencing at the amino terminal) :
  • the pres- ent invention provides polypeptides characterized by in ⁇ cluding part or all of the following amino acid sequence (commencing at its amino terminal) :
  • the above-deseribed sequence also conforms with sparse published data on avian growth hormone, and is specifically an amino acid sequence for avian growth hormone native to the turkey species.
  • the above amino acid sequences for avian growth hormones of the type native to chicken and turkey species may be directly expressed by selected host microorganisms with an initial methionine residue at the amino terminal, i.e., as the [Met ] analog of the mature polypeptide or expressed as a portion of a fusion protein from which they may be isolated by selective cleavage. They may also be directly expressed with an approximately 25 amino acid "leader" region, set out below, which is duplicative of a leader region synthesized in avian (chicken and turkey) pituitary cells and which is apparently processed off of the mature polypeptide prior to entry of the growth hormone into circulation. -20 NH 2 -Met Ala Pro Gly Ser Trp Phe Ser Pro Leu Leu He Ala
  • Polypeptides provided by the present invention may suitably be employed in the manufacture of thera ⁇ Chamberic or growth-inducing agents useful in treating and raising fowl, especially chickens and turkeys, or other animals.
  • the polypeptides also provide a large and reliably-produced quantity of pure avian growth hormone substances useful in the development of information concerning avian metabolism.
  • purified and isolated polypeptides of the invention are expected to be highly useful in immunological determinations wherein they may serve as readily quantified antigens or stimulants for the development of specific antibodies to avian growth hormones.
  • Polypeptides of the invention are produced in quantity by recombinant methods wherein selected host cells, preferably of a microorganism such as E.coli or yeast, are transformed with a hybrid viral or plasmid DNA vector including a specific DNA sequence coding for the polypeptide or polypeptide analog and the polypeptide is synthesized in the host upon transcription and transla ⁇ tion of the DNA sequence.
  • selected host cells preferably of a microorganism such as E.coli or yeast
  • a hybrid viral or plasmid DNA vector including a specific DNA sequence coding for the polypeptide or polypeptide analog
  • the polypeptide is synthesized in the host upon transcription and transla ⁇ tion of the DNA sequence.
  • the present invention provides puri ⁇ fied and isolated continuous, double-stranded DNA se ⁇ quences capable of directing the synthesis in a selected host microorganism of a polypeptide possessing one or more of the biochemical and immunological properties of an avian growth hormone.
  • Two illustrative DNA sequences of the invention include the following sequence of nucle ⁇ otide bases in the top strand thereof, 5'-ACC TTC CCT GCC ATG CCC CTC TCC AAC CTG TTT GCC AAC - GCT GTG CTG AGG GCT CAG CAC CTC CAC CTC CTG GCT GCC GAG ACA TAT AAA GAG TTC GAA CGC ACC TAT ATT CCG GAG GAC CAG AGG TAC ACC AAC AAA AAC TCC CAG GCT GCG TTT TGT TAC TCA GAA ACC ATC CCA GCT CCC ACG GGG AAG GAT GAC GCC CAG CAG AAG TCA GAC ATG GAG CTG CTT CGG TTT TCA CTG GTT CTC ATC CAG TCC TGG CTC ACC CCC GTG CAA TAC CTA AGC AAG GTG TTC ACG AAC AAC TTG GTT TTT GGC ACC TCA GAC AGA GTG TTT
  • DNA sequences for avian growth hormone polypeptides may be incorporated into a vector in association with a DNA sequence coding for an amino acid leader sequence such as is apparently present in "precursor" polypeptides formed in avian pituitary cells, and/or with 5* and 3 1 untranslated sequences.
  • sequence may be employed to supply unique endonuclease enzyme restriction sites to facilitate insertion into a selected vector.
  • sequence may be employed to supply unique endonuclease enzyme restriction sites to facilitate insertion into a selected vector.
  • 5 lowing 5' untranslated DNA sequence and leader sequence may be employed 5' to the codon for the initial amino acid in one of the avian growth hormone sequences above:
  • one or more transcription stop codons or part or all of the "naturally occurring" 3' 5 untranslated DNA sequences may be employed 3' to the codon for the carboxy terminal (He 191) amino acid.
  • the above-speci ied DNA sequences by virtue of their cDNA origins (i.e., by virtue of development as a complement to chicken growth hormone mRNA and turkey growth hormone mRNA, respectively) , comprise DNA triplet codons especially suited for high level expression of growth hormone in avian cells.
  • polypeptide expres ⁇ sion in microorganisms such as bacteria and yeast
  • the inven ⁇ tion thus comprehends the above-specified sequences and alternative sequences of nucleotide bases comprising triplet codons specifying polypeptides with the same sequence of amino acids.
  • a specific aspect of the invention is the provision of DNA sequences which code for polypep- tides displaying the biochemical and immunological prop- erties of avian growth hormone and which comprise nucle- * otides arranged entirely or in part according to such codon usages as are endogenous to avian cells and/or entirely or in part according to codons noted to be the subject of preferred or "optimal" expression in micro ⁇ organisms.
  • One such composite or “hybrid” sequence of the invention includes a cDNA-derived duplicate of a chicken growth hormone nucleotide sequence wherein approximately 32 of the initial codons (commencing at the 5' end of the polypeptide coding region) are replaced and supplemented by the following manufactured DNA sequence:
  • GCC GCT GAA ACT TAT AAA
  • GAA TT 3' CGG CGA CTT TGA ATA TTT CTT AAGC 5'
  • This exemplary composite sequence is seen to provide various features making it especially useful in securing microbial expression of polypeptides of the invention in that its manufactured portion includes: a base codon specifying an initial methionine (base pair numbers 8-10) ; base codons optimally expressed in bacteria; and bases facilitating use of readily available restriction endonucleases in the formation of hybrid transformation vectors (an Xbal "sticky end") .
  • DNA sequences of the invention may also be entirely manufactured (and substan ⁇ tially include only host-preferred codons) according to the synthetic methods of co-owned, co-pending U.S. Patent Application Serial Nos. 375,494 (filed May 6, 1982) and 483,451 (filed April 15, 1983) by.Alton, et al.
  • the DNA sequences of the invention are inserted into microorganism trans ⁇ formation vectors such as self-replicating, selectable plasmids derived from E.coli plasmid pBR322.
  • the hybrid plasmids are employed to transform suitable host micro ⁇ organisms, such as E.coli K-12.
  • the transformation vectors in addition to including the DNA sequence capable of directing the synthesis of a polypeptide possessing one or more of the biochemical and immunolog- ical properties of avian growth hormone, additionally may comprise natural or synthetic transcription promotor/ regulator DNA sequences 5' to the polypeptide coding region.
  • an avian growth hormone polypeptide substance comprising: (1) transforming a selected microorganism with a transforma ⁇ tion vector including a DNA sequence capable of directing microorganism synthesis of a polypeptide possessing one or more of the biochemical and immunological properties of avian growth hormone; (2) growing microorganisms so transformed under suitable nutrient conditions; and, (3) isolating from said microorganisms the product of the expression of said DNA sequence therein.
  • a DNA vector, designated CGH-T21, suitable for use in practice of the invention to stably transform bacterial cells and to thereby allow for the expression therein of readily isolatable quantities of a polypep- tides according to the invention has been deposited with the American Type Culture Collection, Rockville, Mary ⁇ land.
  • the plasmid vector is harbored in a collection of E.coli C600 cells given A.T.C.C. deposit No. 39182.
  • a high molecular weight polypeptide having the biochemical and immunological properties of avian growth hormone native to the chicken species has been produced in isolatable quantities in cells other than those of functional avian pituitary tissue.
  • No. 39182 which includes a replica of the cDNA; the trans ⁇ formation of cells with the vector to secure expression of the desired polypeptide; and,, testing of polypeptide products of transformed cells.
  • Also involved in the making of the present invention was the development of an illustrative, alternative DNA sequence including, in part, a manufactured DNA sequence including E.coli pref ⁇ erence codons for the desired polypeptide; the develop ⁇ ment of an alternative expression vector allowing for higher levels of bacterial expression of desired polypep ⁇ tides; and a series of manipulations performed on turkey pituitary tissue and resulting in isolation of cDNA for avian growth hormone of turkey species origins.
  • Chicken pituitary glands were extracted from one-year-old spent hens and immediately frozen in liquid nitrogen. Approximately 0.4 grams of chicken pituitary (roughly 40-50 pituitaries) were extracted using the guanidinium thiocynate procedure for the quantitative isolationof intact RNA. [Chirgwin, et al.. Biochemistry, 18, pp. 5294-5299 (1979)]. Briefly, the Chirgwin, et al. procedure in ⁇ volves homogenizing the chicken pituitaries in a stock solution of guanidinium thiocynate, a strong denaturant.
  • the homogenates are centrifuged to sediment particulate material and the supernates are mixed with acetic acid and incubated to precipitate the nucleic acid.
  • the material is then centrifuged and the resulting pellet resuspended in guanidine hydrochloride stock solution.
  • the RNA is re-precipitated in solution with acetic acid and ethanol, incubated, and centrifuged. After a final re-precipitation from guanidine hydrochloride, the re-precipitated material is centrifuged again and the pellets dispersed in ethanol at room temperature. Fol ⁇ lowing centrifugation, the ethanol is removed from the pellet by a stream of nitrogen and the RNA dissolved in sterile water.
  • RNA is reprecipitated with sodium acetate (pH 7.0) and ethanol.
  • RNA is sedimented from the ethanol suspension by centrifugation, the pellets thor ⁇ oughly washed with ethanol, dried with nitrogen, and dissolved again in sterile water.
  • the final solution in sterile water contains the purified extracts of total RNA from the chicken pituitaries.
  • the RNA solution is passed through a column containing oligodeoxythy idylate [oligo(dT)] (Collaborative Research, Inc.).
  • oligodeoxythy idylate oligo(dT)
  • Example 1 The following example is directed to construc ⁇ tion of cDNA clones from the messenger RNA isolated in Example 1.
  • Example 1 Prior to its use in the cDNA synthesis proce ⁇ dure of Okayama, et al.. Molecular s Cellular Biology, 2_, pp. 161-170 (1982) , the poly-A + messenger RNA isolated in Example 1 was pre-treated with methylmercury hydrox ⁇ ide (Alpha Ventron) at a final concentration of 4 mM for 5 minutes at room temperature.
  • methylmercury hydrox ⁇ ide treatment denatures interactions of messenger RNA, both with itself and with contaminating molecules that inhibit translation. See, Payvar, et al., J.Biol.Chem. , 258, pp. 7636-7642 (1979).
  • a vector primer is prepared by digesting the plasmid reco binant pBR322-SV40 (map units 0.71-0.86) (Paul Berg, Ph.D., Stanford University) with Kpnl (Bethesda Research Labs) endonuclease which cuts the circular plasmid at one site. After the DNA is extracted, deoxythymid late (dT) residues are added to the Kpnl endonuclease generated termini of the plasmid with calf thymus terminal deoxynucleotidyl transferase (Enzo Biochemicals) .
  • the DNA, now having poly-T tails, is then digested with Hpal (Bethesda Research Labs) endo ⁇ nuclease which removes one poly-T tail.
  • Hpal Bethesda Research Labs
  • the large DNA fragment which contains the origin of pBR322 DNA replica- tion and one poly-T tail is purified by agarose gel electrophoresis, and absorption and elation from an - oligodeoxyaden late [oligo (dA) ] cellulose column (Collaborative Research, Inc.).
  • oligodeoxyguan l e, [oligo (dG) ]-tailed linker DNA is prepared by digesting pBR322-SV40 (map units 0.19-0.32) (Berg) with PstI endonuclease (AMGen) which cuts the circularized plasmid at two restriction sites, thereby generating a large DNA fragment and a small DNA fragment. Tails of deoxyguanylate residues are then added to each end of the two fragments, with termi ⁇ nal deoxynucleotidyl transferase.
  • the extracted and precipitated DNA is then digested with Hindlll endo ⁇ nuclease (Bethesda Research Labs) , which cuts the restric ⁇ tion site on the smaller fragment creating two fragments of unequal size.
  • Hindlll endo ⁇ nuclease Bethesda Research Labs
  • the smallest oligo (dG) -tailed linker is then purified by agarose gel electrophoresis.
  • the first step in cDNA synthesis involves adding the methylmercury hydroxide-treated mRNA to the reverse transcription reaction, which contains 2-mercapto ethanol, T ⁇ s, magnesium chloride, 32P- -dCTP
  • the plasmid vector DNA functions as the primer for the synthesis of the first cDNA strand.
  • Annealing of the poly-A messenger RNA to the poly-dT-tailed vector DNA generates the substrate for reverse transcription of the mRNA sequence.
  • the double-stranded plasmid- cDNA:mRNA from the reverse transcription reaction is added to a mixture containing 32P- ⁇ -dCTP and terminal deoxynucleotidyl transferase.
  • This step adds oligo (dC) tails to the 3' ends of the vector primer c-DNA hybrid.
  • the pellet from the oligo (dC) addition reaction is then added to a reaction containing Hindlll endonuclease.
  • Hindlll endonuclease digestion removes the oligo (dC) tail from the vector primer DNA terminus by cleavage at the unique Hindlll restriction site near that end.
  • the mRNA:cDNA hybrid being a very poor substrate for Hindlll endonuclease, remains intact during digestion, with limiting quantities of the restriction enzyme.
  • the Hindlll endonuclease digested oligo-(dC)- tailed cDNA:mRNA plasmid is incubated in a mixture con ⁇ taining the oligo (dG)-tailed linker DNA.
  • Addition of E.coli ligase (New England Biology Labs) mediates cycli- zation by a covalent joining of the linker and vector DNAs via their Hindlll cohesive ends and a non- ⁇ ovalent base paired join made to the cDNA:mRNA duplex via the oligo (dG) tail of the linker and to the oligo (dC) tail of the cDNA.
  • RNA strand in the resulting double-stranded re ⁇ ombinant is replaced by DNA by reac ⁇ tion with E.coli RNaseH (PL Biochemical Co.) , E.coli DNA ligase, E.coli DNA polymerase-1, and four deoxynucle- oside triphosphates (Sigma) .
  • the RNaseH introduces nicks in the RNA strand, while Pol/I and the four deoxynucle- oside triphosphates replace the RNA segments by nick translation.
  • E.coli DNA ligase joins the newly- synthesized DNA fragments into a continuous second cDNA strand.
  • the oligo (dG) tail of linker DNA serves as the primer for copying any unpaired deoxyribosylcytidine (dC) sequence and extending the strand to the cDNA region.
  • dC deoxyribosylcytidine
  • the full or nearly full-length reverse transcript of the mRNAs are preferentially converted to duplex cDNAs.
  • the cDNAs are then transformed by incuba ⁇ tion into a host microorganism E.coli K-12 strain HB101 for amplification.
  • OMPI The following example is directed to isolation - of chicken species avian growth hormone-coding (here ⁇ after "cGH") clones by hybridization of pBR322-SV40 recombinants containing the cDNA sequence with a nick- 5 translated probe comprising DNA fragments coding for bovine growth hormone and rat growth hormone, respec ⁇ tively.
  • cGH avian growth hormone-coding
  • the complexity of the mRNA isolated from chicken pituitaries necessitates screening a moderate number of recombinant clones containing cDNA inserts in order to locate the clone which codes for synthesis of a 5 polypeptide having one or more of the biochemical and im unological properties of chicken growth hormone.
  • bacteria containing recombinants with cDNA inserts are spread on a nitrocellulose filter o (Millipore) laid on a agar plate. The plate is then in ⁇ cubated to establish small colonies which are replicated to another nitrocellulose filter. The replicas are incu ⁇ bated until distinct colonies appear.
  • the bacteria on the filters are lysed on sheets of Whatman 3 MM paper barely saturated with sodium hydroxide (0.5M). After blotting, the process is repeated again with sodium hydroxide, tris (1M) , and tris (0.5M)-sodium chloride (1.5M).
  • the recombinant clones obtained are hybridized with either a radiolabelled 490 base pair PvuII fragment from pBR322-bGHl5 (a plasmid including a bovine growth
  • OMPI hormone gene or a radiolabelled 816 base pair fragment * from pRGH-1 [Seeberg, et al. Nature, 270, p. 486 (1977)] , a plasmid including a rat growth hormone gene.
  • plasmid fragments containing the bovine growth hormone c DNA sequence and the rat growth hormone DNA sequence, respectively when hybridized with the recombinants con ⁇ taining chicken pituitary cDNA inserts, detect the homolo ⁇ gous chicken growth hormone DNA sequence.
  • the probe fragments align with the corresponding cDNA chicken 0 growth hormone sequence in several recombinants. Eight clones containing the cGH sequence were detected using autoradiography.
  • the following example is directed to charac ⁇ terizing by sequence analysis a cGH-coding clone.
  • the single-stranded DNA phage M-13 was used as a cloning vector for supplying single-stranded DNA tem ⁇ plates from the double-stranded cDNA clones.
  • the Sanger, et al. method revealed the sequence for the cGH protein 5 coding DNA single strand in M-13, which included the cGH coding region having a single BamHI recognition site; an - approximately 75 base sequence 5' to the cGH coding region which sequence presumptively codes for a 25 amino acid leader region; and 3 1 and 5* untranslated regions. 5 This sequence is set out in Table I below with its accom ⁇ panying amino acid translation:
  • Example 5 relates to con- struction of an expression vector.
  • the following example is directed to con ⁇ structing a plasmid expression vector, cGH-T21 harbored in A.T.C.C. 39182 cells.
  • a pBR322-derived expression plasmid (Pint- ⁇ - tx B4) was used which contained a tryptophan promoter (trp) sequence and an Xbal site 3' to a Shine/Delgarno sequence followed by a structural gene coding for another polypeptide.
  • the extraneous polypeptide was excised and the cGH gene was inserted by a process involving deletion from the sequence set out in Example 4 of the 5' untrans- lated region and a 78 base pair 5' region coding for the
  • CGH-T21 was transformed into E.coli K-12 strain C600 (A.T.C.C. 39182) for expression analysis.
  • the following example is directed to transform ⁇ ing E.coli with the plasmid vector to secure expression ooff tthhee ddeessiirreedd ppoollypeptide (i.e., [Met ,des-Thr ]cGH) in bacterial cells.
  • ooff tthhee ddeessiirreedd ppoollypeptide i.e., [Met ,des-Thr ]cGH
  • Cells of E.coli K-12 strain c600 (A.T.C.C. 39182) containing the cGH expression plasmid cGH-T21 are grown in M9 minimal salts supplemented with 0.4% glucose, 1 mM MgSO., 0.1 mM CaCl 2 / 5 mg/ml casamino acids and 10 ⁇ g/ml thiamine in a 37°C shaker to an O.D. (A g00 ) of 1. At this point, 15 ml of cells are treated with 3-Indoleacrylic acid (final cone. 10 ⁇ g/ml) and allowed to continue to shake at 37°C for 1 hour.
  • 3-Indoleacrylic acid final cone. 10 ⁇ g/ml
  • 10 ml cells are then pelleted at approximately 4,000xg in a Beckman J-6 and resuspended in 0.5 ml of a Tris-sucrose-lysozyme buffer (Goeddel, et al.. Nature, 281, 544-548 [1981]) ' containing, in addition, 0.05% sodium dodecyl sulfate and 1 mM phenylmethylsulfonylfluoride. The cells are then placed on ice for 30 minutes after which they are treated with 0.11 ml of a DNase buffer and 10 ⁇ g of
  • the supernate is then spun at 12,000xg for 15 minutes in an Eppendorf microfuge and the supernate is decanted into a tube containing 25 ⁇ l of 10% Triton X-100.
  • the sample is then stored on ice until it is subjected to an RIA as described in Example 8.
  • the following example relates to biochemical similarity of icrobially-produced chicken species avian growth hormone according to the invention and growth hormone purified from chicken pituitary glands.
  • the MW of the microbially-produced avian growth hormone of the invention was determined by
  • CSR603 harboring plasmid, CGH-T21.
  • the pro ⁇ cedure involves growing lOmls of CSR603 (cGH-T21) in K-media [Rupp, et al. , J.Mol.Biol. , 61, pp. 25-44 (1971)] g to a concentration of 2 x 10 cells/ml. 5 ml of cells are then irradiated in a 60 mm petri dish on a shaker platform with a UV dose of 1-5 J/m . The cells are then allowed to shake for one hour at 37°C, at which point cycloserine is added at 100 ⁇ g/ml. The cultures remain shaking for an additional 8-12 hours.
  • Cells are col ⁇ lected by centrifugation and washed twice with Herschey salts [(Worcel, et al. , J.Mol.Biol. , 82, pp. 91-105 (1974)]. Cells are resuspended in 2.5 ml of Herschey media and incubated at 37°C with aeration for one hour. One milliliter of cells is then placed in a 50 ml poly-
  • a second protein of MW 24,000 is seen only in cultures containing the plasmid (cGH-T21) encoding the cGH gene.
  • the MW of the microbially-produced avian growth hormone is thus similar to that found for the natural growth hormone isolated from chicken pituitary tissue.
  • the following example describes a sequential competition radioimmunoassay employing the avian growth hormone polypeptide produced in cells transformed with CGH-T21.
  • Example 6 The bacterial lysate of Example 6 including chicken growth hormone expressed in E.coli cells upon transformation with plasmid CGH-T21 was assayed using a sequential competition radioimmunoassay [Zettner, et al., Clin.Chem., 2£, pp. 5-14 (1979)].
  • oGH ovine growth hormone
  • the appropriate immune complexes are formed.
  • the fixed amount of la- belled oGH added to the reaction mixture complexes with any anti-oGH or anti-cGH not previously bound to the bacterial lysate cGH competitor.
  • the amount of radio ⁇ activity in the resulting precipitate is inversely pro ⁇ portional to the amount of cGH competitor present in the sample.
  • This immunoassay thus indicates that the bac ⁇ terial lysates of Example 6 do, indeed, contain the DNA sequence capable of synthesizing a polypeptide exhib ⁇ iting one or more of the biological characteristics of chicken growth hormone including the ability of the poly ⁇ peptide to form antigen-antibody complexes with anti ⁇ bodies to homologous and heterologous species growth hormone.
  • a nucleotide sequence coding for the first third 5 of the gene has been synthesized jji vitro as a "replace ⁇ ment" for approximately the initial one-third of the cDNA-derived gene.
  • the purpose of the synthesis is to place an Xbal site sticky end three nucleotides upstream of an introduced ATG [Met ] transcription start site, 0 to have an Eco RI site upstream from (and partially over ⁇ lapping) the naturall -occurring Ta I site, and to have a TaqI site sticky end for splicing the gene back to ⁇ gether at the naturally occurring TaqI site.
  • the syn ⁇ thesis is also undertaken to obtain optimal codon usage
  • nucleotide sequence 15 for expression in E.coli and have an adenine- and thiamine-rich region for the first few amino acids of the sequence. Because of the frequency of prolines and alanines in the sequence, however, it is difficult to get an AT-rich start sequence.
  • CAAACGCTGT TCTGCGTGCA CAGCATCTGC ATCTGCTGGC CGCTGAAACT GTTTGCGACA AGACGCACGT GTCGTAGACG TAGACGACCG GCGACTTTGA 1 0
  • the above sequence may be 35 inserted in plasmid CGH-T21 (from which an Xbal/TaqI fragment has been excised to provide for microbial expression of [Met ,des-Thr ]cGH polypeptide.
  • the synthetic fragment is manufactured as previously described with a threonine- specifying codon adjacent (3 1 ) to the ATG codon speci ⁇ fying methionine.
  • the following example relates to manipulations leading to development of another expression vector successfully employed to secure high levels of expression of a polypeptide having the biochemical and immunological properties of avian growth hormone, specifically [Met ,des-Thr ]cGH polypeptide.
  • plasmid pCFM414 (A.T.C.C. 40076) includes a temperature-sensitive mutation in the copy control region. Upon transformation with this vector, host cell cultures grown at 30°C will have a low number of copies of the plasmid. The plasmid copy number increases fifty- fold or more (i.e., "runs away") within the host cells upon elevation of the culture temperature above 34 ⁇ C. Growth at 37°C will ordinarily be lethal to the cells but prior to cell death there is an opportunity for multiple transcriptions of plasmid DNA.
  • Plasmid CGH-T21 (in A.T.C.C. 39182) was digested with restriction endonucleases EcoRI and Ncol, thereby
  • OMPI excising a fragment containing a trp promoter originally present in Pint- ⁇ -txB4 followed by the cGH polypeptide coding region and a few base pairs from the untranslated
  • Plasmid pCFM414 was digested with EcoRI and EcoRI.
  • the following example describes procedures for obtaining and sequencing DNA for turkey species avian growth hormone.
  • tGH turkey growth hormone
  • the single-stranded DNA phage M-13 was used as a cloning vector for supplying single-stranded DNA tem- plates from the double-stranded cDNA clones.
  • T.o et al. method revealed the sequence for the tGH protein ' coding DNA single strand in M-13.
  • This sequence like that of cGH, includes the polypeptide coding region con ⁇ taining a single BamHI recognition site; an approximately 75 base pair sequence 5 1 to the protein coding region, which sequence presumptively codes for a 25 amino acid leader region; and 3* and 5' untranslated regions.
  • the polypeptide-coding DNA sequence is set out in Table II below with its accompanying amino acid sequence:
  • threonine rather than alanine is specified at position number 11; methionine rather than valine is specified at position 0 number 90; arginine rather than lysine is specified at position number 144; serine rather than asparagine is specified at position number 151; and asparagine rather than threonine is specified at position number 190.
  • the amino acids specified for the "leader" sequence are iden- 5 tical except for the presence of glycine at position 4, rather than glutamine.
  • the tGH coding region should be provided with an initial ATG codon 3' to the codon for the amino terminal threonine residue and the sequence inserted in a transformation vector at a site under control of a suitable promoter/regulator DNA se ⁇ quence.
  • An expression vector similar to that constructed in Example 5 for cGH can be employed to express tGH in a microbial host.
  • a "replacement" nucleotide sequence for the first third of the tGH-derived gene may be con ⁇ structed and employed as in Example 9 to place selected restriction endonuclease enzyme recognition sites in the sequence, to obtain optimal codon usage for expression in E.coli, and to have an adenine- and thiamine-rich start sequence.
  • yeast expression for example, would involve vector development in the context of the requirements peculiar to the synthesis of protein in yeast host cells, e.g., use of DNA vectors capable of autonomous replication in yeast, use of yeast promoter/regulator regions, and the like. See, e.g., Valenzuela, et al. , Nature, 298, pp. 347-350 (1982) re ⁇ lating to expression of hepatitis B surface antigen in yeast cells.
  • polypeptide products of the invention are expected to have commercial utility in animal husbandry procedures involving, e.g., continuous
  • OMPI low level delivery of products to poultr as a feed or water supplement or as active components delivered by means of a slow dissolving (or slow ingredient-releasing) subcutaneous implant Such delivery systems would be particularly advantageous in treatment of young fowl during early growth stages.

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Abstract

Nouveaux polypeptides possédant les propriétés biochimiques et immunologiques de l'hormone de croissance d'aviens sont obtenus par des procédés d'ADN recombinants. On décrit des nouvelles séquences d'ADN qui peuvent guider la synthèse de ces polypeptides dans des micro-organismes hôtes sélectionnés. Dans un mode préférentiel de réalisation, un nouveau polypeptide ayant les propriétés de l'hormone de croissance des aviens provenant de la poule est produit, résultant de l'expression bactérienne d'un nouveau plasmide, cGH-T21. Ce plasmide est logé dans des cellules transformées d'E. coli C600 enregistrées sous le numéro de dépôt 39182 auprès de l'A.T.C.C. (American Type Culture Collection). On décrit également une séquence d'ADN pouvant guider la synthèse d'un nouveau polypeptide ayant les propriétés de l'hormone de croissance des aviens provenant des dindons.
PCT/US1983/001388 1982-09-16 1983-09-13 Hormone de croissance d'aviens Ceased WO1984001150A1 (fr)

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
US4828987A (en) * 1984-05-14 1989-05-09 Merck & Co., Inc. Avian retrovirus-bovine growth hormone DNA
US5759816A (en) * 1984-08-27 1998-06-02 Bio-Technology General Corp. Expression vectors containing λPL promoter and T1 T2 rRNA termination sequence plasmids containing the vectors hosts containing the plasmids and related methods
CN1072720C (zh) * 1992-05-09 2001-10-10 株式会社乐喜 鸡生长激素基因及其在大肠杆菌中的表达

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US4710473A (en) * 1983-08-10 1987-12-01 Amgen, Inc. DNA plasmids

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US4342832A (en) * 1979-07-05 1982-08-03 Genentech, Inc. Method of constructing a replicable cloning vehicle having quasi-synthetic genes
US4362867A (en) * 1980-12-10 1982-12-07 Research Corporation Recombinant cDNA construction method and hybrid nucleotides useful in cloning
US4366246A (en) * 1977-11-08 1982-12-28 Genentech, Inc. Method for microbial polypeptide expression

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IE48385B1 (en) * 1978-08-11 1984-12-26 Univ California Synthesis of a eucaryotic protein by a microorganism
US4273875A (en) 1979-03-05 1981-06-16 The Upjohn Company Plasmid and process of isolating same
US4293652A (en) 1979-05-25 1981-10-06 Cetus Corporation Method for synthesizing DNA sequentially
US4269731A (en) 1979-08-27 1981-05-26 Argus Chemical Corporation Antimony mercaptocarboxylic acid ester stabilizers for polyvinyl chloride resin compositions and process
IE52755B1 (en) * 1980-08-26 1988-02-17 Univ California Bovine pre-growth and growth hormone

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US4366246A (en) * 1977-11-08 1982-12-28 Genentech, Inc. Method for microbial polypeptide expression
US4342832A (en) * 1979-07-05 1982-08-03 Genentech, Inc. Method of constructing a replicable cloning vehicle having quasi-synthetic genes
US4362867A (en) * 1980-12-10 1982-12-07 Research Corporation Recombinant cDNA construction method and hybrid nucleotides useful in cloning

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4828987A (en) * 1984-05-14 1989-05-09 Merck & Co., Inc. Avian retrovirus-bovine growth hormone DNA
US5759816A (en) * 1984-08-27 1998-06-02 Bio-Technology General Corp. Expression vectors containing λPL promoter and T1 T2 rRNA termination sequence plasmids containing the vectors hosts containing the plasmids and related methods
CN1072720C (zh) * 1992-05-09 2001-10-10 株式会社乐喜 鸡生长激素基因及其在大肠杆菌中的表达

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JPS59501852A (ja) 1984-11-08
EP0106170A2 (fr) 1984-04-25

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